Functional Synthetic Models of Cu-dependent Monooxygenases

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Project Summary: In this research project, we develop synthetic inorganic copper complexes to understand the fundamental aspects of structure and function in Cu-dependent monooxygenase enzymes. These metalloenzymes contain 1 or 2 Cu ions in their active center and they couple the reduction of O2 with the oxidation of substrates via formation of transient Cun/O2 species. We are particularly interested in studying the reactivity of mononuclear Cu/O2 intermediates since they have been proposed as active oxidants in the hydroxylation of strong C-H bonds in enzymes such as particulate methane monooxygenases (pMMOs) and lytic polysaccharide monooxygenases (LPMOs). Many questions concerning the identity of the active Cu/O2 species remain unanswered, including: i) oxidation state of Cu (CuI vs. CuII vs. CuIII); ii) reduction/protonation state of O2 (O2−,(H)O22−, (H)O2−) and the pKa and redox potentials associated with these Cu/O2 species; iii) mechanism by which the Cu/O2 intermediates carry out C-H hydroxylations (e.g. O-O cleavage mechanism before or after C-H oxidation?; generation of high-valent Cu-oxyl species before substrate hydroxylation?). In this research proposal, we tackle this problem using two different approaches: 1) We utilize ligand scaffolds (L) that contain C-H substrates covalently attached to their structure (substrate- ligands) that permit us to generate and characterize LCu/O2 species and evaluate their reactivity towards intramolecular C-H hydroxylation. Substrate-ligand modifications will permit us to: i) evaluate the ability of the Cu/O2 species to oxidize sp3 C-H bonds and sp2 C-H bonds; ii) control the stereo-electronic properties of the Cu complexes by the use of different ligand donors (i.e. N2, N3, N4) that will lead to the generation of mononuclear and dinuclear LCu/O2 species, and analyze their reactivity towards intramolecular C-H hydroxylation including characterization of reaction intermediates, kinetics and computations; iii) utilize this approach (Cu-directed hydroxylations) to develop synthetic protocols to promote challenging organic transformations such as enantioselective C-H hydroxylations and one-pot synthesis of 1,3-oxazines. 2) We synthesize mononuclear Cu complexes bearing redox-active ligands with tunable H-bonds that stabilize Cu-hydroxo and Cu-oxyl cores. These unusual Cu complexes are able to reach multiple oxidation states via oxidation of the metal and/or ligand scaffold. These high-valent CuO(H) cores will be characterized by various spectroscopic methods and their ability to perform biorelevant intermolecular 2e− C-H hydroxylations will be examined systematically using the Bordwell equation (i.e. species with higher redox potential and higher pKa should be capable of oxidizing stronger C-H bonds), kinetic experiments and analysis of the reactions products derived from hydroxylation (e.g. organic product(s) and oxidation/protonation state of the final Cu complexes). Overall, these studies will contribute to a broader understanding of the biochemical role of Cu ions involved in O2 reduction and biologically relevant oxidations.